Receptor-mediated endocytosis of biofilm-forming Enterococcus faecalis by rat peritoneal macrophages

Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics. Studies were taken up to identify virulence factors and to characterise pathogenic mechanisms of such infections to evaluate potential targets for treatments alternative to antibiotic therapy. This study was carried out to evaluate the contribution of extracellular polysaccharide expressed by Enterococcus faecalis to resistance to phagocytosis and survival within rat peritoneal macrophages.

Six E. faecalis clinical isolates were tested for their ability to survive within rat peritoneal macrophages. Cytochalasin D, colchicine and monodansylcadaverine were used to investigate the route of enterococcal entry inside macrophages.

Four of the isolates were able to produce extracellular polysaccharide and form biofilm after growth in glucose-supplemented medium, while no production could be detected in glucose deficient medium. Two isolates were polysaccharide-negative in both conditions. Isolates expressing extracellular polysaccharide were able to survive for more than 24 h compared to polysaccharide-negative bacterial cells of the same strain grown in glucose-deficient medium, which were readily cleared. Cytochalasin D virtually abolished the number of viable intracellular bacteria, after growth in either trypticase soy broth (TSB) or TSB supplemented with glucose; colchicine and monodansylcadaverine strongly affected survival of polysaccharide-positive bacteria, significantly more than that of polysaccharide-negative ones.

Biofilm-forming E. faecalis survived within rat peritoneal macrophages significantly better than polysaccharide-negative isolates. Perturbators of cytoskeleton and of surface receptors turnover, indicated receptors-mediated endocytosis as the most likely route for enterococcal entry into macrophages.